Although previous studies from this group have indicated the absolute requirement for reverse transcriptase (RT)-associated ribonuclease H (RNase H) activity for virus replication, this enzymatic function has received little attention as an antiviral target. However, the finding that HIV-1 RNase H and integrase (IN) are derived from the same superfamily of nucleotidyl transferases has allowed the implementation of screening strategies based on IN inhibitors previously identified at the NCI. Several natural products have been identified that act on both RNase H and IN, while a limited number show selectivity for the individual proteins. A patent for the use of these compounds has recently been submitted. In collaboration with J. Beutler (NCI), M. Hawkins (NCI), and M. Parniak (University of Pittsburgh), we are now screening several small-molecule libraries of synthetic and natural compounds (~250,000 in total) for antagonists of HIV-1 RNase H. The current project applies a high-throughput fluorescence-based RNase H assay for primary screening. Once complete, secondary screens will be initiated to (a) eliminate molecules quenching fluorescence without impairing enzyme function, (b) exclude nucleic acid-binding agents, (c) determine the selectivity of RNase H inhibitors with respect to the human counterpart, (d) evaluate inhibitor potency, using substrates mimicking specialized RNase H-mediated events, and (e) assess in vivo potency and cytotoxicity. Candidate antagonists will be evaluated by a combination of biochemical and biophysical approaches. Finally, combinatorial chemical synthesis will be undertaken in collaboration with the University of Pittsburgh Center for Combinatorial Chemistry.